REMOVAL OF PROTEOGLYCANS INCREASES EFFICIENCY OF RETROVIRAL GENE-TRANSFER

We have previously shown that medium conditioned by virus producer cel ls inhibits retrovirus transduction, and that a portion of the inhibit ory activity is sensitive to chondroitinase ABC. In this study, we hav e quantitatively evaluated the fraction of the inhibitory activity tha t is due to chondroitinase ABC-sensitive material and partially charac terized the inhibitors. The kinetics of chondroitinase ABC digestion o f glycosaminoglycans and virus inhibitory activity in cell culture med ium were measured, and the results used to estimate the amount of the chondroitinase ABC-sensitive virus inhibitory activity that was initia lly in the medium. We found that up to 76% of the inhibitory activity of medium conditioned by packaging cells derived from NIH 3T3 cells is sensitive to chondroitinase ABC. The remainder of the inhibitory acti vity is not sensitive to other glycosaminoglycan lyases (heparitinase I or heparinase I), which suggests that substances other than glycosam inoglycans or proteoglycans are present in virus stocks and inhibit tr ansduction. To further characterize the inhibitors, proteoglycans from conditioned medium were purified by batch anion exchange and size exc lusion chromatography. Two major size groups (100 kDa and 950 kDa) of proteoglycans were isolated. Transduction was inhibited 50% by 0.6 mu g/mL of the high-molecular-weight proteoglycan or by 1.7 mu g/mL of th e low-molecular-weight proteoglycan. Significantly, the proteoglycans, because of their large size and poor sieving properties, coconcentrat ed with virus particles concentrated by ultrafiltration and prevented any significant increases in transduction efficiency. Transduction eff iciencies of virus stocks were increased more than tenfold by ultrafil tration, but only when the concentrated virus was treated with chondro itinase ABC. (C) 1998 John Wiley & Sons, Inc.